Project Summary/Abstract: Targeted therapies that inhibit oncogenic kinases such as EGFR, ALK, ROS1 (TKIs) have significantly improved the survival of lung cancer patients whose tumors harbor activating genomic alterations in these genes. Unfortunately, these therapies are not curative and patients eventually develop drug resistance leading to disease progression. Recent work by our group and others has demonstrated that genomic mechanisms of acquired drug resistance can evolve from residual drug-tolerant cells that that initial survive drug treatment and then subsequently acquire the genomic alteration during therapy. This suggests that treatment strategies that can alter the survival and evolution of residual tumor cells may pre-empt the emergence of resistant clones. In preliminary studies using preclinical models of EGFR mutant and ALK fusion lung cancer, we have observed that cancer cells surviving initial TKI treatment exhibit increased expression and activity of the APOBEC3A cytidine deaminase. Additionally, analysis of clinical tumor samples from EGFR and ALK lung cancer patients revealed accumulation of APOBEC mutations during sequential TKI therapy. We hypothesize that APOBEC3A- mediated DNA and RNA editing induced by TKI treatment facilitates the emergence of drug tolerant clones in lung tumors during treatment and facilitates the evolution of acquired drug resistance. In this project, we will develop novel biochemical and computational tools to study APOBEC3A mutagenesis in EGFR mutant and ALK fusion non-small cell lung cancer experimental models and clinical tumor specimens. These studies will establish a causal link between TKI treatment and induction of APOBEC mutagenesis. Next, we will investigate whether activation of innate immune signaling pathways in response to genomic instability and/or expression of RNA repeat elements is necessary and sufficient for TKI-induced APOBEC3A mutagenesis. Finally, we will determine whether induction of APOBEC3A promotes the survival of drug tolerant clones and subsequent development of acquired drug resistance. These studies will yield fundamental biological insights into how TKI resistance evolves in oncogene-addicted cancers and identify new vulnerabilities that can be targeted to delay or prevent resistance from developing.